Source: Polymerase chain reaction
Four pahses for PCR
2n / cycle
Reference DOI: 10.1100/tsw.2011.124.
| Roche | Bio-Rad | Applied Biosystems | Qiagen |
|---|---|---|---|
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LightCycler®480 |
CFX96 TouchTM |
StepOneTM |
Rotor-Gene Q |
| Gene detection | Gene expression |
|---|---|
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Number of publications in PubMed related to qPCR.
(Number of publications in PubMed related to specific qPCR applications.)
Reference DOI: 10.1016/j.bdq.2014.08.002
Summary of the research articles published in different journals.
The data was retrieved from Scopus by using the key word “Real-Time PCR for Food Analysis”
Reference DOI: 10.1007/s13197-016-2205-0
Non-specific dsDNA binding dyes
Sequence-specific fluorescence detection chemistry
Reference DOI: 10.1038/sj.leu.2402922
Reference DOI: 10.1002/9780470027318.a1406.pub2
Reference DOI: 10.1038/sj.leu.2402922
Reference DOI: 10.1007/s13197-016-2205-0
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線上 primer 設計 http://bioinfo.ut.ee/primer3-0.4.0/ |
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線上 primer 設計 http://www.premierbiosoft.com/qOligo/Oligo.jsp?PID=1 |
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檢查目標產物結構 http://unafold.rna.albany.edu/?q=mfold/download-mfold |
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多重序列比對 https://www.ebi.ac.uk/Tools/msa/clustalo/ Reference DOI: 10.1002/pro.3290 |
曾經有過這樣的設計 (142-232),看起來好像不錯!?
結果,因為立體結構的關係,使得擴增效率非常差!
Reference DOI: 10.1016/j.bdq.2014.08.002
Cp calculation
Abosolute
Relative
Tm calling analysis (Melting curve analysis)
Picture source:What Is A Cycle Threshold (Ct) Value In qPCR?
\(N = N_{0}\times E^{Cp}\)
\(LogN = LogN_{0}+Cp\times LogE\)
\(Cp=-(\frac{1}{LogE})LogN_{0}+\frac{LogN}{LogE}\)
Reference DOI: 10.1002/9780470027318.a1406.pub2
Reference DOI: 10.5114/aoms.2014.44871
\(Relative\ ratio = \frac{Target\ gene\ concentration}{Reference\ gene\ concentration}\)
Reference gene:
Reference DOI: 10.1016/j.bbrc.2003.11.177 (被引用1,367次)
Optional:
\(Calibrator\ Normalize\ Ratio = \frac{\frac{Target\ gene\ concentration}{Reference\ gene\ concentration}(Sample)}{{\frac{Target\ gene\ concentration}{Reference\ gene\ concentration}(Calibrator)}}\)
Calibrator:
Correct the differences in detection sensitivity between Target / Reference gene and several experiments (e.g., long-term studies)
Not treated cell line
Sample before experiment
Reference: Roche 教育訓練專員講義
Reference DOI: 10.1016/j.mcp.2010.04.002
Minimum Information for Publication of Quantitative Real-Time PCR Experiments, (2009)
限定使用Roche原廠耗材!
(盤子與封模,唯一限定的材料)